Posttranslational modification of eukaryotic proteins, particularly therapeutic proteins such as immunoglobulins, is often necessary for proper protein folding and function. Because standard prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in production of these therapeutic proteins. Yeast and fungi are attractive options for expressing proteins as they can be easily grown at a large scale in simple media, which allows low production costs, and yeast and fungi have posttranslational machinery and chaperones that perform similar functions as found in mammalian cells. Moreover, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells as well as more complex eukaryotic cells such as mammalian or insect cells (De Pourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31). Despite these advantages, many therapeutic proteins are still being produced in mammalian cells, which produce therapeutic proteins with posttranslational modifications most resembling the native human proteins, whereas the posttranslational modifications naturally produced by yeast and fungi often differ from those found in mammalian cells.
To address this deficiency, new strains of yeast and fungi are being developed that produce posttranslational modifications that more closely resemble those found in native human proteins. More specifically, news strains of yeast and fungi have been genetically modified so that they express recombinant proteins having N-glycan patterns resembling that of native human proteins. The general strategies include the elimination of endogenous glycosylation enzymes that are involved in producing high mannose N-glycans (such as och1p or Alg3p in yeast), and the introduction of certain glycosyltransferases in order to reproduce the sequential reaction steps of the mammalian glycosylation pathway, including α1,2 mannosidase, GnTI, mannosidase II, GnTII, GalT, SiaT enzymes (Wildt and Gerngross, 2005, Nature, 3: 119-127; De Pourcq et al., 2010, Appl Microbiol Biotechnol, 87:1617-1631).
Mammalian and human cells express fucosyltransferase (FucTs) activities and FucTs are therefore one of the enzyme families of interest for remodeling N-glycan patterns on the surface of recombinant glycoproteins produced in yeast or fungi. The presence of fucosylated structures on glycoproteins has indeed been shown to be advantageous in some cases. More specifically, in the production of monoclonal antibodies, immunoglobulin and related glycoproteins comprising Fc fragment, it is well known that the presence of fucosylated N-glycans influence antibody dependent cytotoxicity (ADCC) activity. In some cases, it is desirable to produce antibodies with fucosylated N-glycans, in order to decrease ADCC activity of the resulting protein. It is further well known that the presence of fucosylated N-glycans influence antibody dependent cellular phagocytosis (ADCP) activity (Shibata-Koyama et al. 2009. Exp Hematol 37:309-21). In some cases, it is desirable to produce antibodies with fucosylated N-glycans, in order to decrease ADCP activity of the resulting protein.
WO 2008/112092 reports materials and methods for making lower eukaryotic expression systems that can be used to produce recombinant, fucosylated glycoproteins. The genetic modification of yeast P. pastoris strain capable of producing glycoproteins that include fucose is described, including the insertion of polynucleotides encoding human GDP-mannose-4,6 dehydratase (GMD), human GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (FX) and human α1,6 fucosyltransferase (FUT8) proteins in such host cell.
Other reports have suggested genetic modification of yeast strains to produce the GDP-L-fucose, the substrate of fucosyltransferase (Chigira et al. 2008, Glycobiology 18 no. 4 pp 303-314; Jä rvinen et al. 2001, Eur J Biochem 268, 6458-6464), and/or human-like fucosylated glycoforms (Ma et al., 2006, Glycobiology 16(12) pp 158-184, US 20050170452, US 2010137565, US2010062485).
Reports of fungal cell expression systems expressing human-like fucosylated N-glycans are lacking. Indeed, due to the industry's focus on mammalian cell culture technology for such a long time, the fungal cell expression systems such as Trichoderma are not as well established for therapeutic protein production as mammalian cell culture and therefore suffer from drawbacks when expressing mammalian proteins. In particular, a need remains in the art for improved filamentous fungal cells, such as Trichoderma fungus cells, that can stably produce heterologous proteins with mammalian-like N-glycan patterns, preferably at high levels of expression.
The invention now provides fungal cell expression system, more specifically Trichoderma cells, or related species such as Neurospora, Myceliophtora, Fusarium, Aspergillus, Penicillium and Chrysosporium species, having reduced protease activity and capable of expressing fucosylated glycoproteins, for example with mammalian-like complex fucosylated N-glycans.